The present invention relates to:
microspheres having a diameter xe2x89xa70.1 and  less than 1xcexc, comprising a biocompatible polysaccharide polymer and optionally at least one active ingredient,
pharmaceutical compositions containing said microspheres administrable by oral, nasal, pulmonary, vaginal or rectal route,
the use of microspheres having a diameter ranging from 0.1 to 1xcexc as carriers for the preparation of pharmaceutical compositions for human genic therapy, for the preparation of diagnostics and in the agroalimentary industry,
a process for the preparation of microspheres having a dimension of between 0.1 and 1xcexc comprising the precipitation of said polymer induced by means of a supercritical antisolvent (SAS).
Major advances have recently been made in pharmaceutical technology to research new methods for the preservation of the intrinsic activity of polypeptides and to render them absorbent. Formulations able to ensure a reproducible absorption of these active molecules have the advantage of lacking side effects, unlike synthetic polymers. Of all the most widely used natural polymers, the category of acidic polysaccharides is of particular interest. One of these, hyaluronic acid, a polysaccharide widely distributed throughout animal organisms, is constituted by units of D-glucuronic acid and N-acetyl D-glucosamine in alternate order. Its molecular weight can vary according to the methods used for its extraction and/or purification (EP 0138572 reg. on Jul. 25, 1990: EPA 0535200 published on Apr. 7, 1993; PCT Application No. WO 95/04132 published on Feb. 9, 1995; PCT Patent Application No. WO 95/24497 published on Sep. 14, 1995).
Besides the polymer""s chemical-physical properties, the release methods and systems for biologically active molecules are also particularly important, such as microspheres which seem to be among the most versatile release systems. EPA 0517565 discloses a process for the preparation of microspheres, whose dimensions range between 1-100 xcexcm, wherein the polysaccharide ester dissolved in an aprotic solvent such as DMSO, is added to a mixture of a high-viscosity mineral oil containing a non ionic surface active agent and ethyl acetate, which is a solvent for DMSO and the mineral oil, but not for the polysaccharide ester, which therefore precipitates in the form of microspheres having therefore the above mentioned dimensions.
Today, various techniques are known which involve the use of supercritical fluids for the production of finely subdivided particles with a narrow granulometric distribution curve. The supercritical antisolvent process is generally performed at moderate temperatures and enables the solvent to be completely removed from the precipitation environment. The applications concern substances that are heat-sensitive or difficult to handle, such as explosives (Gallagher, P. M. et al., 1989, Supercritical Fluid Science and Technologyxe2x80x94Am. Chem. Soc. 334-354). Other applications concern the production of polymers in the form of fibers (Dixon, D. J. et al, 1993, J. Appl. Polym. Sci. 50, 1929-1942) and in the form of microparticles, including microspheres (Dixon, D. J., et al., 1993, AIChE J., 39, 1, pp 127-139). In the pharmaceutical field, the main interest is in the treatment of proteins (Tom, J. W., et al, 1994, Supercritical Fluid Engineering Science, pp 238-257, ACS Symp. Chap. 19, Ed. H. Kiran and J. F. Brennecke; Yeo, S. D., et al, 1993, Biotech. and Bioeng., 41, pp 341-346) and biodegradable polymers, such as poly(L-lactic acid) (Randolph, T. W., et al, 1993, Biotechnol. Prog., 9, 429-435; Yeo, S. D., et al, 1993, Macromolecules, 26, 6207-6210). Various methods have been devised for precipitation with a supercritical antisolvent. The semi-discontinuous method (Gallagher et al., 1989), involves injection of the antisolvent in the liquid solution which has already been prepared in the desired working conditions. The operation must be performed in a stepwise fashion to ensure that the liquid is removed, the final quantities of product are very limited and the spheres measure far more than 1xcexc in size.
Precipitation with a compressed antisolvent (PCA) involves injection of the solution in the high-density supercritical fluid (SCF) (Dixon et al., 1991; Dixon and Johnston, 1993). The injection times are much reduced to guarantee complete dissolution of the liquid, so the quantity of precipitate is very low, giving microfibers with an ordered structure.
The continuous process (Yeo et al., 1993a) enables the solution and the antisolvent to be injected simultaneously in the precipitation environment: the liquid expands and evaporates in the continuous phase, constituted by the SCF. The solution is injected through a micrometric nozzle with a diameter ranging between 10 and 30xcexc. Solutions must be diluted to avoid blocking the nozzle and to present reticulate structures being formed. Consequently, the quantity of solid solute injected is very low. Moreover, a high ratio between the volume of antisolvent and solution must be used to continuously remove the liquid solvent from the precipitation vessel.
When the solution is placed in the precipitator and the container is loaded by means of SCF up to the desired pressure, the process assumes a completely discontinuous character (Yeo et al., 1993 a,b). By this technique, microspheres with a diameter of over 1xcexc have been obtained. All the methods described here are accompanied by a final washing step to prevent the precipitate being resolubilized by the solvent. However, none of the cited techniques has been specifically applied to the production of high-molecular-weight biocompatible polysaccharide polymers and in particular the HYAFFs, namely the ester of hyaluronic acid, which are obtained by the procedure described in U.S. Pat. No. 4,851,521.
The Applicant has unexpectedly found that with the discontinuous SAS technique it is possible to obtain in quantitative yields microspheres with a diameter of less than 1xcexc comprising an ester of a biocompatible acidic polysaccharide polymer, selected from the group consisting of: hyaluronic acid esters, crosslinked esters of hyaluronic acid, esters of chitin, esters of pectin, esters of gellan, esters of alginic acid.
Object of the present invention are therefore microspheres having a dimension xe2x89xa70.1xcexc and  less than 1xcexc comprising a biocompatible polysaccharide polymer.
A further object of the present invention are pharmaceutical compositions administrable by oral, nasal, pulmonary, vaginal or rectal route, containing said microspheres as vehicling agents or carriers in combination with at least one active ingredient and optionally with further conventional excipients.
A further object of the present invention relates to said microspheres further comprising at least one of the following active principles: a pharmaceutically active polypeptide, a Granulocyte Macrofage Colony Stimulating Factor (GMCSF), a trophic factor, an immunoglobulin, a natural or a synthetic derivative of a ganglioside, an antiviral, an antiasthmatic an antiinflammatory agent, an antibiotic and an antimycotic agent.
A further object of the present invention relates to pharmaceutical compositions administrable by oral, nasal, pulmonary, vaginal or rectal route containing the microspheres inglobating the above mentioned active principles, optionally in combination with other conventional excipients.
A further object of the present invention relates to the use of said microspheres as carriers in the preparation of diagnostics and in agroalimentary industry. Moreover the microspheres having a diameter ranging from 0.1 to 1xcexc containing a biocompatible acidic polysaccharide ester selected from the group consisting of: hyaluronic acid esters, esters of chitin, esters of pectin, esters of gellan, esters of alginic acid can be advantageously used as vehicling agent or carriers of a gene, for the preparation of pharmaceutical compositions for the treatment of diseases associated with genic defects.
A further object of the present invention resides in the discontinuous process for the preparation of microspheres having a dimension comprised between 0.1 and 1xcexc and comprising the precipitation of said polymer induced by means of a supercritical antisolvent (SAS). The process object of the present invention comprises the following steps:
a) dissolving the polysaccharide biocompatible polymer in an aprotic solvent at concentrations ranging from 0.1 to 5% by weight,
b) charging the solution of step (a) in a pressure proof container having at the top and at the base steel filters with an average cut-off lower than 0.1xcexc;
c) loading from underneath the antisolvent until reaching the pressure at which said fluid becomes supercritical at a temperature ranging from 25 to 60xc2x0 C.,
d) removing the aprotic solvent, by flowing said supercritical fluid,
e) depressurizing the pressure proof container and collecting the precipitated product.
Contrarily to what one could foresee from the above mentioned prior art (teaching that, with the SAS discontinuous technique, process times are longer than with the continuous one, nucleation occurs in the bulk liquid phase where the supercritical antisolvent is dissolved and therefore the formation of large particles with broad granulometric distribution is expected), surprisingly the expanding conditions adopted with the process according to the present invention enable the onset of the nucleation process in a well-expanded media so that the formation of a high number of nucleation centres is achieved. This factor, combined with the amorphous nature of the solid solute, leads to the formation of microspheres whose dimension is comprised in the above mentioned range and moreover with a narrow granulometric distribution curve.